MiP2005: Session 5

Mitochondrial Physiology Network 10.9: 63 (2005) - download pdf


Regulation of cytochrome c oxidase kinetics in vivo by phosphorylation of subunits via different signal cascades in bovine heart and isolated rat cardiomyocytes.

Sebastian Vogt, P Weber, A Rhiel, B Kadenbach

Cardiovascular Research Laboratory, Biomedical Science Center, Philipps-University of Marburg, Germany. - vogts@med.uni-marburg.de 

    The control of respiration by the availability of ADP is generally explained by Peter Mitchells chemiosmotic hypothesis via the mitochondrial membrane potential. A new control of respiration by ATP, independent of the membrane potential, was found by Arnold and Kadenbach [1], based on binding of ATP to subunit IV of cytochrome c oxidase (COX). High cellular ATP/ADP ratios induce an allosteric inhibition of COX activity, which, however, is only found with the phosphorylated enzyme. The allosteric ATP-inhibition was postulated to keep the mitochondrial membrane potential ∆ψm at low levels, thus preventing the formation of reactive oxygen species (ROS) [2]. Lee et al. [3] described recently a correlation between cAMP-dependent  phosphorylation of Tyr304 of subunit I and the allosteric ATP-inhibition of COX in liver. We investigated the effect of wortmannin (inhibitor of phosphatidylinositol-3 kinase, PI3K), PP2 (inhibitor of non-receptor tyrosine kinases) and IBMX (inhibitor of phosphodiesterases) on ATP-inhibition of COX and phosphorylation of subunit II in bovine tissue. Ground bovine heart was incubated at 4 °C under shaking with cell culture medium and corresponding additions. From the incubated tissue mitochondria were isolated and suspended in 25 mM phosphate buffer containing 0.1 % Tween 20 and protease- and phosphatase-inhibitors. The kinetics of ascorbate respiration was measured polarographically at increasing concentrations of cytochrome c in the presence of 5 mM ADP as well as 5 mM ATP with an ATP-regenerating system (PEP and PK). Wortmannin and PP2 turned off the ATP-inhibition, whereas IBMX stimulated it. While PP2 completely prevented phosphorylation of subunit II, wortmannin and IBMX had little effect on its phosphorylation. This was shown by Western Blot with an antibody against phosphotyrosine after immunoadsorption of the holoenzyme from laurylmaltoside-solubilized mitochondria with a monoclonal antibody against subunit IV. From the results we conclude that the ATP-inhibition of COX could be turned on by phosphorylation via 3 different signal cascades: (1) via the G-receptor/cAMP pathway, (2) via the PI3K/Akt pathway, and (3) via a nonreceptor tyrosine kinase pathway. Corresponding experiments were performed with isolated adult rat cardiomyocytes, obtained by Langendorff-perfusion with collagenease. Tyrosine-phosphorylation of subunit IV was found to be turned off completely by incubation of cardiomyocytes with wortmannin, accompanied by abolition of ATP-inhibition of COX.

1.  Arnold S, Kadenbach B (1997) Cell respiration is controlled by ATP, an allosteric inhibitor of cytochrome c oxidase. Eur. J. Biochem. 249: 350-354.

2.  Lee I, Bender E, Arnold S, Kadenbach B (2001) New control of mitochondrial membrane potential and ROS-formation. Biol. Chem. 382: 1629-1636.

3.  Lee I, Salomon AR, Ficarro S, Mathes I, Lottspeich F, Grossman LI, Hüttemann M (2005) cAMP-dependent tyrosine phosphorylation of subunit I inhibits cytochrome c oxidase activity. J. Biol. Chem. 280: 6094-6100.

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